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Abstract:

From antibodies that can be used to immunostain atherosclerotic tissue
sections, the present inventors selected antibodies applicable to in vivo
imaging, and analyzed their specificities. The result showed that
fluorescently labeled anti-oxidized LDL/β2GPI complex
antibodies that are specific to a particular epitope were effective for
imaging.

Claims:

1. An antibody of any one of (a) to (e) below, which binds to a complex
of oxidized LDL and β2-glycoprotein I (oxidized
LDL/β2GPI complex): (a) an antibody comprising a heavy chain
that comprises CDR1 having the amino acid sequence of SEQ ID NO: 2, CDR2
having the amino acid sequence of SEQ ID NO: 3, and CDR3 having the amino
acid sequence of SEQ ID NO: 4; (b) an antibody comprising a heavy chain
that comprises a heavy-chain variable region having the amino acid
sequence of SEQ ID NO: 1; (c) an antibody comprising a light chain that
comprises CDR1 having the amino acid sequence of SEQ ID NO: 7, CDR2
having the amino acid sequence of SEQ ID NO: 8, and CDR3 having the amino
acid sequence of SEQ ID NO: 9; (d) an antibody comprising a light chain
that comprises a light-chain variable region having the amino acid
sequence of SEQ ID NO: 6; and (e) an antibody that comprises a pair of
the heavy chain of (a) or (b) above and the light chain of (c) or (d)
above.

2. An antibody that binds to the same epitope as the antibody of any one
of claim 1.

3. The antibody of claim 1, which is a humanized or chimeric antibody.

4. An imaging agent for visualizing an arteriosclerosis site, which
comprises an antibody that binds to a complex of oxidized LDL and
β2-glycoprotein I (oxidized LDL/β2GPI complex).

5. An imaging agent for visualizing an arteriosclerosis site, which
comprises the antibody of claim 1.

6. The imaging agent of claim 5, for determining the location and/or size
of atheroma in arteriosclerosis.

7. An imaging kit for visualizing an arteriosclerosis site, which
comprises an antibody that binds to a complex of oxidized LDL and
β2-glycoprotein I (oxidized LDL/β2GPI complex).

8. An imaging kit for visualizing an arteriosclerosis site, which
comprises the antibody of claim 1.

9. A method of screening for a candidate compound for a therapeutic agent
for arteriosclerosis, which comprises the steps of: (a) administering a
candidate compound to an arteriosclerosis model nonhuman animal
administered with the antibody of claim 1; (b) carrying out imaging of an
arteriosclerotic plaque in an arteriosclerosis model nonhuman animal
administered with the candidate compound and in an arteriosclerosis model
nonhuman animal not administered with the candidate compound; (c)
comparing the size or location of an arteriosclerotic plaque between the
arteriosclerosis model nonhuman animal administered with the candidate
compound and the arteriosclerosis model nonhuman animal not administered
with the candidate compound; and (d) selecting a candidate compound that
reduces or eliminates an arteriosclerotic plaque in the arteriosclerosis
model nonhuman animal administered with the candidate compound as
compared to the arteriosclerosis model nonhuman animal not administered
with the candidate compound.

10. An imaging agent for visualizing an arteriosclerosis site, which
comprises an antibody that binds to a complex of oxidized LDL and
β2-glycoprotein I (oxidized LDL/β2GPI complex), and
which comprises a pair of heavy chain described in (a) or (b) below and
light chain described in (c) or (d) below: (a) an antibody comprising a
heavy chain that comprises CDR1 having the amino acid sequence of SEQ ID
NO: 2, CDR2 having the amino acid sequence of SEQ ID NO: 3, and CDR3
having the amino acid sequence of SEQ ID NO: 4; (b) an antibody
comprising a heavy chain that comprises a heavy-chain variable region
having the amino acid sequence of SEQ ID NO: 1; (c) an antibody
comprising a light chain that comprises CDR1 having the amino acid
sequence of SEQ ID NO: 7, CDR2 having the amino acid sequence of SEQ ID
NO: 8, and CDR3 having the amino acid sequence of SEQ ID NO: 9; (d) an
antibody comprising a light chain that comprises a light-chain variable
region having the amino acid sequence of SEQ ID NO: 6.

11. The imaging agent of claim 10, which is an in vivo imaging agent.

12. The imaging agent of claim 10 for in vivo administration.

13. An imaging agent for visualizing an arteriosclerosis site, which
comprises the antibody of claim 1.

14. Use of the antibody of claim 1 for the manufacture of an imaging
agent for visualizing an arteriosclerosis site.

15. The antibody of claim 1 for use in an imaging method for visualizing
an arteriosclerosis site.

Description:

TECHNICAL FIELD

[0001] The present invention relates to antibodies against an oxidized
LDL/β2GPI complex, and non-invasive diagnostic methods for
arteriosclerosis using the antibodies such as methods for identifying
atherosclerotic lesion sites and methods for monitoring the therapeutic
effects.

BACKGROUND ART

[0002] Diagnostic methods for assessing the condition of arteriosclerosis,
which have already been put to practical use, include, for example, the
four methods described below.

[0003] "Ankle-brachial pressure index": When blood pressure is measured at
the arm and ankle levels in the supine position, the ankle blood pressure
is normally slightly higher. However, the narrowing of a blood vessel
reduces the downstream blood pressure, which results in a decrease in the
ratio of ankle blood pressure to brachial blood pressure (ABI). A
decrease in ABI not only indicates arteriosclerosis in the artery of the
lower limb but also suggests systemic arteriosclerosis.

[0004] "Pulse wave velocity test": A method for estimating the progression
of arteriosclerosis by assessing arterial stiffness. In healthy
individuals, blood vessels are elastic and thus vascular walls absorb
vibration, resulting in a reduction in pulse wave velocity. As
arteriosclerosis advances, the wave velocity increases. Thus, the
progression of arteriosclerosis can be estimated using the velocity as an
indicator.

[0005] "Carotid ultrasound examination": A method for estimating the
progression of systemic arteriosclerosis by observing carotid arteries
which run very close to the surface of skin and have an interior
condition that is easy to observe by ultrasound.

[0006] "MR angiography (MRA)" and "CT angiography (CTA)": Angiography was
used as a major diagnostic imaging method for vascular diseases, but
image information that is almost comparable to angiography but obtained
in a less invasive manner has become available. The advantages of CTA
include: (1) high spatial resolution; (2) simple examination; and (3)
superiority in detecting calcified lesions.

[0007] The above-described "ankle-brachial pressure index" and "pulse wave
velocity test" can neither identify the site of atherosclerosis nor
diagnose the progression at each site. Thus, these methods only provide
indirect scores to assess arteriosclerosis.

[0008] Unlike pulse wave velocity test or such, "carotid ultrasound
examination" is superior in that it enables direct graphical observation
of the inside of blood vessels. However, the condition of vascular wall
is assessed based on the contrasting density and shape in ultrasonic
images, and thus clinicians and laboratory technicians who conduct the
test are required to have skills. Furthermore, the test cannot identify
the site of atherosclerosis or diagnose the progression at individual
sites in blood vessels other than the carotid artery.

[0009] Meanwhile, methods for monitoring the progression of
arteriosclerosis include ELISA systems for measuring the oxidized
LDL/β2GPI complex in blood (Japanese Patent Nos. 3370334 and
3898680; WO2003/022866, WO2004/023141). However, conventional ELISA for
measuring the oxidized LDL/β2GPI complex can be used to
estimate the size but not the site of atherosclerotic plaque.

[0010] Meanwhile, even when MRI or radiolabeled imaging is used, the
condition of vascular wall is assessed based on the contrasting densities
and shapes in ultrasonic images, and thus clinicians and laboratory
technicians who conduct the test are required to have skills and
expertise (U.S. Pat. Nos. 6,716,410 and 6,375,925).

[0021] An objective of the present invention is to provide antibodies
against an oxidized LDL/β2GPI complex, and non-invasive
diagnostic methods for arteriosclerosis using the antibodies such as
methods for identifying arteriosclerosis lesion sites and methods for
monitoring the therapeutic effects.

Means for Solving the Problems

[0022] From antibodies that can be used to immunostain atherosclerotic
tissue sections, the present inventors selected antibodies applicable to
in vivo imaging, specifically to visualize atherosclerotic plaques, in
particular, the location and size of atheroma in the body. Then, the
prevent inventors analyzed the specificities of the antibodies. The
result showed that fluorescently labeled anti-oxidized
LDL/β2GPI complex antibodies that are specific to a particular
epitope were effective for imaging.

[0023] Specifically, the present invention provides:

[1] an antibody of any one of (a) to (e) below, which binds to a complex
of oxidized LDL and β2-glycoprotein I (oxidized
LDL/β2GPI complex):

[0027] (d) an antibody comprising a light chain that comprises a
light-chain variable region having the amino acid sequence of SEQ ID NO:
6; and

[0028] (e) an antibody that comprises a pair of the heavy chain of (a) or
(b) above and the light chain of (c) or (d) above;

[2] an antibody that binds to the same epitope as the antibody of any one
of [1]; [3] the antibody of [1] or [2], which is a humanized or chimeric
antibody; [4] an imaging agent for visualizing an arteriosclerosis site,
which comprises an antibody that binds to a complex of oxidized LDL and
β2-glycoprotein I (oxidized LDL/β2GPI complex); [5]
an imaging agent for visualizing an arteriosclerosis site, which
comprises the antibody of any one of [1] to [3]; [6] the imaging agent of
[4] or [5], for determining the location and/or size of atheroma in
arteriosclerosis; [7] an imaging kit for visualizing an arteriosclerosis
site, which comprises an antibody that binds to a complex of oxidized LDL
and β2-glycoprotein I (oxidized LDL/β2GPI complex);
[8] an imaging kit for visualizing an arteriosclerosis site, which
comprises the antibody of any one of [1] to [3]; [9] a method of
screening for a candidate compound for a therapeutic agent for
arteriosclerosis, which comprises the steps of:

[0029] (a) administering a candidate compound to an arteriosclerosis model
nonhuman animal administered with the antibody of any one of [1] to [3];

[0030] (b) carrying out imaging of an arteriosclerotic plaque in an
arteriosclerosis model nonhuman animal administered with the candidate
compound and in an arteriosclerosis model nonhuman animal not
administered with the candidate compound;

[0031] (c) comparing the size or location of an arteriosclerotic plaque
between the arteriosclerosis model nonhuman animal administered with the
candidate compound and the arteriosclerosis model nonhuman animal not
administered with the candidate compound; and

[0032] (d) selecting a candidate compound that reduces or eliminates an
arteriosclerotic plaque in the arteriosclerosis model nonhuman animal
administered with the candidate compound as compared to the
arteriosclerosis model nonhuman animal not administered with the
candidate compound;

[10] an imaging agent for visualizing an arteriosclerosis site, which
comprises the antibody of any one of [1] to [3]; [11] use of the antibody
of any one of [1] to [3] for the manufacture of an imaging agent for
visualizing an arteriosclerosis site; and [12] the antibody of any one of
[1] to [3] for use in an imaging method for visualizing an
arteriosclerosis site.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033]FIG. 1 is diagrams showing antibody reactivities to immobilized
antigen. The antibodies were monoclonal antibodies obtained by immunizing
BALB/c mice with the oxidized LDL/β2GPI complex as an antigen.
The horizontal axis indicates antibody concentration, and the vertical
axis indicates the absorbance.

[0035] FIG. 3 is graphs showing a test of competitive inhibition by
antigen. The horizontal axis indicates antigen concentration in a liquid,
and the vertical axis indicates the percent inhibition (%) determined
when taking the absorbance in the absence of inhibitory antigen as 100%.
3H3 and 4C12 are antibodies that recognize β2GPI bound to
oxidized LDL. These antibodies do not recognize free β2GPI.
2H6, 3D4, and 2A12 are antibodies reactive to free β2GPI.

[0037]FIG. 5 is photographs showing fluorescent immunostaining of the
aortic valve in arteriosclerosis-prone model mice (apoE.sup.-/- fed a
high fat diet). The photographs show results of fluorescent
immunostaining using other antibodies against the oxidized
LDL/β2GPI complex. Antibodies positive for atheroma in the
staining were only antibodies 3H3 and A.

[0038]FIG. 6 is photographs showing IVIS 200 fluorescence imaging using
specific antibody (reflection fluorescence microscopy). In vivo:
ApoE.sup.-/- mice were fed a high fat diet for six months or more.
Imaging agents were administered to the mice at the caudal vein. After
two to 24 hours, in vivo fluorescence was observed and photographed under
inhalation anesthesia using IVIS 200. The ApoE.sup.-/- mice were observed
after shaving, because their black hair absorbs fluorescence. Ex vivo:
Mice euthanized were thoracotomized. The heart and aorta were exposed,
and a small incision was made in the right auricular appendage. Then, a
needle was inserted into the left ventricle and the heart was perfused
with 10 ml of cold PBS. The heart and aorta were excised and their
reflection fluorescence microscopic images were recorded using IVIS 200.

[0039] FIG. 7 is photographs showing IVIS 200 fluorescence imaging
(excitation, 640 nm; emission, 720 nm). Experiment 1: physiological
saline (PBS; control), Cy5.5-labeled antibody A, or Cy5.5-labeled
antibody 3H3 was administered at the caudal vein to apoE.sup.-/- mice fed
a high fat diet. Twenty four hours after administration, the mice were
photographed alive for the full-body image after removing their thoracic
skin. Then, the heart intact with thoracic aorta was excised and
photographed. Experiment 2: Hearts and aortae excised from mice
administered with PBS, Cy5.5-labeled antibody 2A12, or y5.5-labeled
antibody 3H3. Administered 3H3 intensely stained the aortic root.
Antibody A also stained to some extent; however, the fluorescence
intensity is weaker as compared to 3H3. There was no stain in the case of
2A12.

[0042] FIG. 10 is a diagram showing fluorescence intensity of Cy5.5 around
the aortic root observed using IVIS 200. The fluorescence intensity was
determined per unit area of the aortic root. The fluorescence of
PBS-administered control mouse was taken as 1.0. When 3H3 was
administered, fluorescence was three times stronger than the control.
When other antibodies were administered, there was no significant change
in the fluorescence intensity.

[0043] FIG. 11 is a diagram showing the amino acid sequence of antibody
3H3. Each CDR is underlined.

MODE FOR CARRYING OUT THE INVENTION

[0044] The present invention provides antibodies that bind to a complex of
oxidatively modified LDL (oxidized LDL) and β2-glycoprotein I
(oxidized LDL/β2GPI complex). The complex is formed between
oxidized LDL and β2GP in atherosclerotic plaques.
β2GP is a serum glycoprotein. The antibodies of the present
invention bind to the complex.

[0045] Specifically, the antibodies of the present invention include those
described below, but are not limited thereto:

[0049] (d) an antibody comprising a light chain that comprises a
light-chain variable region having the amino acid sequence of SEQ ID NO:
6; and

[0050] (e) an antibody comprising a pair of the heavy chain of (a) or (b)
above and the light chain (c) or (d) above.

[0051] The present invention also provides antibodies that bind to the
same epitope as an antibody of the present invention that binds to the
complex of oxidized LDL and β2-glycoprotein I (oxidized
LDL/β2GPI complex). Such antibodies recognize a particular
epitope on the oxidized LDL/β2GPI molecule which is a complex
formed with oxidized LDL.

[0052] Whether an antibody recognizes the same epitope as another antibody
can be confirmed, for example, by their competition for the epitope,
although the test method is not limited thereto. The competition between
antibodies can be assessed by competitive binding assays. The method
includes ELISA, fluorescence resonance energy transfer (FRET), and
fluorometric microvolume assay technology (FMAT®). The amount of a
particular antibody bound to antigen is indirectly correlated with the
binding activity of a competitor antibody candidate (test antibody),
which competes for the binding to the same epitope. Specifically, as the
amount or affinity of a test antibody for the same epitope increases, the
amount of an antibody bound to the antigen decreases, and the amount of
test antibody bound to the antigen increases. More specifically, an
appropriately labeled antibody is added to the antigen together with a
test antibody, and then the bound antibody is detected using the label.
The amount of an antibody bound to the antigen can be readily determined
by labeling the antibody in advance. Such labels are not particularly
limited; however, appropriate labeling methods are selected depending on
the technique. Such labeling methods include, for example, fluorescent
labeling, radiolabeling, and enzyme labeling.

[0053] Herein, "antibody that recognizes the same epitope" refers to an
antibody that can reduce the amount of labeled antibody bound by at least
50%, when a test antibody is used at a concentration typically 100 times
higher, preferably 80 times higher, more preferably 50 times higher, even
preferably 30 times higher, and still preferably 10 times higher than the
IC50 of the non-labeled antibody, where IC50 is defined as a
concentration of a non-labeled antibody at which the amount of the
labeled antibody bound is decreased by 50% due to the binding of the
non-labeled antibody.

[0054] The antibodies of the present invention include both polyclonal and
monoclonal antibodies. Methods for preparing and purifying monoclonal and
polyclonal antibodies are known in the field, and described, for example,
in "Harlow and Lane, Antibodies: A Laboratory Manual (New York: Cold
Spring Harbor Laboratory Press, 1988)".

[0055] The antibodies of the present invention also include recombinant
antibodies such as humanized antibodies and chimeric antibodies.
"Humanized antibody" refers to an antibody whose structure is similar to
that of a human antibody. Such humanized antibodies and chimeric
antibodies include human-type chimeric antibodies (for example,
antibodies in which some portions have been humanized, antibodies whose
CH2 region has been humanized, antibodies whose Fc domain has been
humanized, antibodies whose constant region has been humanized),
CDR-grafted humanized antibodies whose constant and variable regions have
been humanized except their complementarity determining regions (CDRs)
(P. T. Johons et al., Nature 321, 522 (1986)), and completely humanized
antibodies. Improvement methods for enhancing the antigen binding
activity of a CDR-grafted human-type antibody have been developed, which
include: methods for selecting human antibody FRs that are highly
homologous to the mouse antibody, methods for producing highly homologous
humanized antibodies, and methods for substituting amino acids in FR
after grafting mouse CDRs to human antibodies (see U.S. Pat. Nos.
5,585,089, 5,693,761, 5,693,762, and 6180370; EP Nos. 451216 and 682040;
Japanese Patent No. 2828340). Such methods can be used to prepare
CDR-grafted human-type antibodies of the present invention.

[0056] Human-type chimeric antibodies can be produced, for example, by
substituting a human anybody constant region for the constant region of
an above-described antibody having the structure of an H-chain variable
region and/or the structure of an L-chain variable region described
above. Such human antibody constant regions include known human antibody
constant regions. A method for producing human-type chimeric antibodies
is described below as an example.

[0058] The transformants are cultured, and then human-type chimeric
antibodies are isolated from the transformants or culture media.
Antibodies can be isolated or purified by an appropriate combination of
methods such as centrifugation, ammonium sulfate fractionation, salting
out, ultrafiltration, affinity chromatography, ion exchange
chromatography, and gel filtration chromatography.

[0059] Meanwhile, human-type CDR-grafted antibodies can be produced, for
example, by the following method. First, the amino acid sequences of
H-chain and L-chain variable regions of an antibody against a particular
antigen, and nucleotide sequences encoding them are determined by the
methods for producing chimeric antibodies as described above. The amino
acid and nucleotide sequences of each CDR are determined as well.

[0061] Furthermore, FR amino acid sequences to be used also include amino
acid sequences resulting from modification of the amino acid sequence of
a selected human FR, as long as the human-type CDR-grafted antibody
produced from it has the activity of specifically binding to the target
antigen. In particular, when a portion of the amino acid sequence of a
selected human FR is replaced with the amino acid sequence of an FR of
the antibody from which CDR is derived, the resulting antibody is very
likely to retain the antibody properties. The number of amino acids to be
modified is preferably 30% or less in a whole FR, more preferably 20% or
less in a whole FR, and still more preferably 10% or less in a whole FR.

[0062] Next, DNAs encoding H-chain and L-chain variable regions are
designed by combining the above-described CDRs with FRs selected by any
one of the methods described above. Based on this design, DNAs encoding
H-chain variable regions and DNAs encoding L-chain variable regions are
prepared by chemical synthesis, biochemical cleavage/ligation, or the
like. Then, an H-chain expression vector is constructed by inserting into
an expression vector the H-chain variable region-encoding DNA, along with
a DNA encoding an H-chain constant region of human immunoglobulin.
Likewise, an L-chain expression vector is constructed by inserting into
an expression vector the L-chain variable region-encoding DNA, along with
a DNA encoding an L-chain constant region of human immunoglobulin.
Expression vectors include, for example, SV40 virus-based vectors, EB
virus-based vectors, and papilloma virus (BPV)-based vectors, but are not
limited thereto.

[0064] The transformants are cultured, and then human-type CDR-grafted
antibodies are isolated from the transformants or culture media.
Antibodies can be isolated or purified by an appropriate combination of
methods such as centrifugation, ammonium sulfate fractionation, salting
out, ultrafiltration, affinity chromatography, ion exchange
chromatography, and gel filtration chromatography.

[0065] Methods for preparing human antibodies are also known. For example,
desired human antibodies with antigen-binding activity can be obtained by
sensitizing human lymphocytes in vitro with an antigen of interest or
cells expressing an antigen of interest; and fusing the sensitized
lymphocytes with human myeloma cells such as U266 (see Japanese Patent
Application Kokoku Publication No. (JP-B) H01-59878 (examined, approved
Japanese patent application published for opposition)). Alternatively,
desired human antibodies can also be obtained by using an antigen of
interest to immunize transgenic animals that have the entire repertoire
of human antibody genes (see International Patent Application WO
93/12227, WO 92/03918, WO 94/02602, WO 94/25585, WO 96/34096, and WO
96/33735).

[0066] In an alternative embodiment, antibodies and antibody fragments can
be isolated from an antibody phage library produced by using the
technique described by McCafferty et al. (Nature, 348: 552-554 (1990)).
Clackson et al. (Nature, 352: 624-628 (1991)) and Marks et al. (J. Mol.
Biol., 222: 581-597 (1991)) reported isolation of mouse and human
antibodies using phage libraries. Subsequently published documents
describe generation of high-affinity (nM range) human antibodies by chain
shuffling (Marks et al., Bio/Technology, 10: 779-783 (1992)); and
combinatorial infection and in vivo recombination as a strategy for
constructing very large phage libraries (Waterhouse et al., Nuc. Acids.
Res., 21: 2265-2266 (1993)). These techniques can serve as an alternative
method for isolating monoclonal antibodies, which are used instead of the
conventional hybridoma method for preparing monoclonal antibodies.

[0067] In this context, the bacteriophage (phage) display is one of the
well-known techniques that enable one to search a large oligopeptide
library and identify library members having the ability to specifically
bind to a target polypeptide. The phage display is a technique that
displays various polypeptides as a fusion protein with the coat protein
on the surface of bacteriophage particles (Scott, J. K. and Smith G. P.
Science 249: 386 (1990)). An advantage of phage display is that it
enables rapid and effective categorization of a large library of
selectively randomized protein mutants (or random cDNA clones) for the
sequences that bind with high affinity to a target molecule. The phage
display of peptide library (Cwirla, S. E. et al., Proc. Natl. Acad. Sci.
USA, 87: 6378 (1990)) or protein library (Lowman, H. B. et al.,
Biochemistry, 30: 10832 (1991); Clackson, T. et al., Nature, 352:624
(1991); Marks, J. D. et al., J. Mol. Biol., 222: 581 (1991); Kang, A. S.
et al., Proc. Natl. Acad. Sci. USA, 88:8363 (1991)) has been used to
screen a vast number of oligopeptides or polypeptides for those that have
a specific binding property (Smith, G P. Current Opin. Biotechnol., 2:668
(1991)). Categorization in a phage library of random mutants requires a
method for constructing and propagating a vast number of mutants; an
affinity purification method using a target receptor; and a method for
assessing the enhanced binding (see U.S. Pat. Nos. 5,223,409, 5,403,484,
5,571,689, and 5663143).

[0069] To date, there are many improved and modified methods developed
based on the basic phage display method. These modifications have
improved the methods for screening peptide or protein libraries based on
a property or ability such as the activity of binding to a selected
target molecule. Recombination means for the phage display method are
described in WO 98/14277. Phage display libraries have been used to
analyze and control bimolecular interactions (WO 98/20169; WO 98/20159)
and properties of constrained helical peptide (WO 98/20036). WO 97/35196
describes a method for isolating affinity ligands, in which bound ligands
are selectively isolated by contacting a phage display library with a
first solution that allows binding of the ligand to a target molecule and
then with a second solution where affinity ligand does not bind to the
target molecule. WO 97/46251 describes a method for isolating high
affinity-binding phages in which a random phage display library is
treated by biopanning using an affinity-purified antibody, followed by
isolation of bound phages, and then by micropanning in the wells of
microplates. There is also a report published on the use of
Staphylococcus aureus protein A as an affinity tag (Li et al., Mol.
Biotech., 9: 187 (1998)). WO 97/47314 describes the use of substrate
subtraction library in identifying enzymatic specificity using a
combinatorial library which may be a phage display library. WO 97/09446
describes a method for selecting enzymes that are suitable as a washing
reagent to be used in phage display. Other methods for selecting proteins
that bind in a specific manner are described in U.S. Pat. Nos. 5,498,538
and 5,432,018, and WO 98/15833. Methods for constructing and screening
peptide libraries are described in U.S. Pat. Nos. 5,723,286, 5,432,018,
5,580,717, 5,427,908, 5,498,530, 5,770,434, 5,734,018, 5,698,426,
5,763,192, and 5,723,323.

[0070] Furthermore, there are known techniques for obtaining human
antibodies by panning with a human antibody library. For example, using a
phage display method, the variable regions of human antibodies can be
expressed as single chain antibodies (scFvs) on the surface of phages to
select phages that bind to an antigen. The DNA sequences encoding the
variable regions of human antibodies that bind to the antigen can be
determined by analyzing the genes of selected phages. When the DNA
sequences of scFvs that bind to the antigen are identified, human
antibodies can be prepared by constructing appropriate expression vectors
carrying these sequences and expressing the antibodies in adequate hosts
introduced with the expression vectors. Such methods are already known
(see WO 92/01047, WO 92/20791, WO 93/06213, WO 93/11236, WO 93/19172, WO
95/01438, and WO 95/15388).

[0071] As an alternative method, the phage display technique (McCafferty
et al., Nature 348: 552-553 (1990)) can be used to produce human
antibodies and antibody fragments in vitro from the immunoglobulin
variable (V) domain gene repertoire of a non-immunized donor. Using this
technique, an antibody V domain gene is cloned in frame with a coat
protein gene of filamentous bacteriophage, for example, M13 or fd, and
then displayed as a functional antibody fragment on the surface of phage
particles. Since filamentous particles contain a single-stranded DNA copy
of the phage genome, screening based on the functional properties of
antibody results in selection of genes encoding an antibody having the
properties. Thus, such phages mimic some characteristics of B cells.
Phage display can be carried out in various modes; see, for example,
Johnson, Kevin S, and Chiswell, David J., Current Opinion in Structural
Biology 3: 564-571 (1993). There are some sources of V gene segments
available for phage display. Clackson et al. (Nature, 352: 624-628
(1991)) have isolated numerous various anti-oxazolone antibodies from a
small random combinatorial library of V genes derived from spleens of
immunized mice. The V gene repertoire of a non-immunized human donor can
be constructed, and antibodies against numerous various antigens
(including self antigens) can be isolated by using the technique
described in either of the following documents without modification:
Marks et al., J. Mol. Biol. 222: 581-597 (1991) or Griffith et al., EMBO
J. 12: 725-734 (1993). See also U.S. Pat. Nos. 5,565,332 and 5,573,905.

[0072] The antibodies of the present invention also include functional
antibody fragments such as Fab, Fab', F(ab')2, Fv, scFv, dsFv,
Diabodies, and sc(Fv)2. Multimers (for example, dimers, trimers,
tetramers, and polymers) of such a functional antibody fragment are also
included in the antibodies of the present invention.

[0073] Fab is a fragment with a molecular weight of about 50,000 that
consists of L-chain and H-chain variable regions, and an H chain fragment
containing CH1 domain and a portion of hinge region. Fab is obtained
by digesting IgG with papain in the presence of cysteine. In the present
invention, an antibody described above can be digested with papain to
prepare such Fab. Alternatively, a DNA encoding a portion of H chain and
the L chain of an antibody described above is inserted into an
appropriate vector. Fab can be prepared from transformants obtained by
transformation using the vector.

[0074] Fab' is a fragment with a molecular weight of about 50,000 obtained
by cleaving the disulfide bond between the H chains of F(ab')2
described below. In the present invention, such F(ab')2 can be
obtained by treating an above-described antibody by pepsin digestion,
followed by cleavage of disulfide bond with a reducing agent.
Alternatively, like Fab, Fab' can be prepared by genetic engineering
using DNA encoding Fab'.

[0075] F(ab')2 is a fragment with a molecular weight of about 100,000
obtained by digesting IgG with pepsin. F(ab')2 is constituted by two
(Fab') fragments linked together via disulfide bond, each of which
consists of L-chain and H-chain variable regions, and an H chain fragment
containing CH1 domain and a portion of hinge region. In the present
invention, F(ab')2 can be prepared by digesting an above-described
antibody with pepsin. Alternatively, like Fab, F(ab')2 can be
prepared by genetic engineering using F(ab')2-encoding DNAs.

[0077] scFv is a single-chain antibody fragment in which the C terminus of
one Fv chain consisting of H-chain and L-chain variable regions is linked
via an appropriate peptide linker to the N terminus of the other Fv
chain. Such peptide linkers include, for example, flexible (GGGGS)3.
For example, a DNA encoding an scFv antibody is constructed using DNAs
encoding the H-chain variable region and L-chain variable region of an
above-described antibody and a DNA encoding a peptide linker, and then
inserted into an appropriate vector. Transformants are obtained by
transformation with the resulting vector. scFv can be prepared from the
transformants.

[0078] dsFv is an Fv fragment whose H-chain and L-chain variable regions
are stabilized with a disulfide bond formed by introducing Cys residues
at appropriate positions in the H-chain and L-chain variable regions. In
each chain, the position at which Cys residue is to be introduced is
determined based on the conformation predicted by molecular modeling. In
the present invention, for example, the conformation is predicted from
the amino acid sequences of H-chain and L-chain variable regions of an
above-described antibody. DNAs are constructed to encode H-chain and
L-chain variable regions that have been introduced with mutations based
on the prediction, and inserted into an appropriate vector. Transformants
are obtained by transformation with the resulting vector. dsFv can be
prepared from the transformants.

[0079] Furthermore, multimers of antibody fragments can be prepared by
linking scFv antibodies, dsFv antibodies, and the like via appropriate
linkers, or fusing them to streptavidin. Fusion antibodies or labeled
antibodies can be prepared from the antibodies (including antibody
fragments) of the present invention by fusing or linking the antibodies
with low molecular weight compounds, proteins, labeling substance, or the
like. Such labeling substances include radioactive substances such as
125I.

[0080] Diabody refers to a bivalent antibody fragment constructed by gene
fusion (Holliger P et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448
(1993); EP 404,097; WO 93/11161). Diabodies are dimers consisting of two
polypeptide chains, where each polypeptide chain has a VL and a VH linked
via a linker short enough to prevent interaction of these two domains,
for example, a linker of about five residues. The VL and VH linked
together in a single polypeptide chain will form a dimer because the
linker between them is too short to form a single-chain variable region
fragment. As a result, the polypeptide chains form a dimer, and thus the
diabody has two antigen binding sites. Diabodies can be prepared by
treating an antibody with an enzyme, for example, papain or pepsin, to
generate antibody fragments, or by constructing DNAs encoding those
antibody fragments and introducing them into expression vectors, followed
by expression in an appropriate host cell (see, for example, Co, M. S. et
al., J. Immunol. 152, 2968-2976 (1994); Better, M. and Horwitz, A. H.,
Methods Enzymol. 178, 476-496 (1989); Pluckthun, A. and Skerra, A.,
Methods Enzymol. 178, 497-515 (1989); Lamoyi, E., Methods Enzymol. 121,
652-663 (1986); Rousseaux, J. et al., Methods Enzymol. 121, 663-669
(1986); Bird, R. E. and Walker, B. W., Trends Biotechnol. 9, 132-137
(1991)).

[0081] sc(Fv)2 is a single-chain minibody produced by linking two VHs and
two VLs using linkers and such (Hudson et al., J. Immunol. Methods 231:
177-189 (1999)). sc(Fv)2 can be produced, for example, by linking
scFvs via a linker.

[0082] The antibodies of the present invention also include fusion
proteins in which an above-described antibody is fused with other
peptides or proteins. The fusion protein can be prepared by linking a
polynucleotide encoding an antibody of the present invention with a
polynucleotide encoding a different peptide or polypeptide in frame, and
introducing this into an expression vector and expressing it in a host.
It is possible to use techniques known to those skilled in the art. Such
a peptide or polypeptide to be fused with an antibody of the present
invention include known peptides, for example, such as FLAG (Hopp, T. P.
et al., BioTechnology 6, 1204-1210 (1988)), 6×His consisting of six
His (histidine) residues, 10×His, influenza hemagglutinin (HA),
human c-myc fragment, VSV-GP fragment, p18HIV fragment, T7-tag, HSV-tag,
E-tag, SV40 T antigen fragment, lck tag, α-tubulin fragment, B-tag,
and Protein C fragment. Furthermore, polypeptides to be fused with an
antibody of the present invention include, for example, GST
(glutathione-S-transferase), HA (influenza hemagglutinin),
β-galactosidase, and MBP (maltose-binding protein).

[0083] The antibodies of the present invention also include antibodies
linked to a labeling substance.

[0085] Fluorescent labels and luminescent labels include those using
enzymatic luminescence (luciferase) and those using fluorescence
(fluorescent proteins such as GFP, DsRed, and Kusabira Orange; and
fluorescent low-molecular-weight substances such as FITC, Cy5.5, and
Alexa Fluor 750).

[0086] When enzymatic luminescence (luciferase) is used, it is necessary
to administer a substrate separately.

[0087] In particular, labels that have reduced influence from the animal's
intrinsic fluorescence, and labels that emit a signal with high skin
permeability are more preferred.

[0088] The present invention also provides DNAs encoding an antibody of
the present invention, vectors inserted with the DNAs, and transformed
cells introduced with the vectors. The vectors include, for example, M13
vectors, pUC vectors, pBR322, pBluescript, and pCR-Script. Alternatively,
when the objective is to subclone and excise cDNAs, the vectors include
pGEM-T, pDIRECT, and pT7, in addition to those described. DNAs encoding
an antibody of the present invention, vectors inserted with the DNAs, and
transformed cells introduced with the vectors are prepared by known
methods.

[0089] DNAs encoding an antibody of the present invention that binds to
the oxidized LDL/β2GPI complex include the following DNAs:

[0094] When an expression vector is used for expression in E. coli, for
example, it should have the above-described characteristics which allow
its amplification in E. coli. Additionally, when the host is E. coli such
as JM109, DH5α, HB101, or XL1-Blue, the vector must have a promoter
that allows efficient expression in E. coli, for example, lacZ promoter
(Ward et al. Nature 341: 544-546 (1989); FASEB J. 6: 2422-2427 (1992)),
araB promoter (Better et al. Science 240:1041-1043 (1988)), or T7
promoter. The vector also includes pGEX-5X-1 (Pharmacia), "QIAexpress
system" (QIAGEN), pEGFP, and pET (for this vector, BL21, a strain
expressing T7 RNA polymerase, is preferably used as the host), in
addition to the above-described vectors.

[0095] Furthermore, the vector may comprise a signal sequence for
polypeptide secretion. When producing proteins into the periplasm of E.
coli, the pelB signal sequence (Lei, S. P. et al. J. Bacteriol. 169: 4379
(1987)) may be used as a signal sequence for protein secretion. The
vector can be introduced into host cell, for example, by the calcium
chloride method or electroporation.

[0097] In order to express proteins in animal cells, such as CHO, COS, and
NIH3T3 cells, the vector must have a promoter necessary for expression in
such cells, for example, SV40 promoter (Mulligan et al. Nature 277:108
(1979)), MMTV-LTR promoter, EF1α promoter (Mizushima et al. Nucleic
Acids Res. 18: 5322 (1990)), CMV promoter, etc). It is even more
preferable that the vector carries a gene for selecting transformants
(for example, a drug-resistance gene that enables discrimination by a
drug (such as neomycin and G418)). Vectors having such characteristics
include, for example, pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.

[0098] In addition, the following method can be used for stable gene
expression and gene amplification in cells: CHO cells deficient in a
nucleic acid synthesis pathway are introduced with a vector (for example,
pCHOI) that carries a DHFR gene which compensates for the deficiency, and
the gene is amplified using methotrexate (MTX). Alternatively, the
following method can be used for transient gene expression: COS cells
whose chromosome contains a gene for expression of SV40 T antigen are
transformed with a vector (such pcD) having an SV40 origin of
replication. It is also possible to use replication origins derived from
polyoma virus, adenovirus, bovine papilloma virus (BPV), or such. To
increase gene copy number in host cells, the expression vectors may
further contain selection markers such as aminoglycoside transferase
(APH) gene, thymidine kinase (TK) gene, E. coli xanthine-guanine
phosphoribosyltransferase (Ecogpt) gene, and dihydrofolate reductase
(dhfr) gene.

[0099] Host cells to be introduced with the vectors are not particularly
limited, and include, for example, E. coli and various types of animal
cells. The host cells can be used, for example, as production systems for
expressing and producing the antibodies of the present invention. The
polypeptide production systems include in vitro and in vivo production
systems. The in vitro production systems include production systems using
eukaryotic or prokaryotic cells.

[0101] The plant cells include, for example, Nicotiana tabacum-derived
cells, which are known as a protein production system and can be cultured
as a callus. The fungal cells include yeasts, for example, the genus
Saccharomyces such as Saccharomyces cerevisiae and Saccharomyces pombe;
and filamentous bacteria, for example, the genus Aspergillus such as
Aspergillus niger.

[0102] When prokaryotic cells are used, production systems using bacterial
cells are available. Such bacterial cells include E. coli, for example,
JM109, DH5a, and HB101, and Bacillus subtilis. The antibodies of the
present invention can be prepared in vitro by culturing cells transformed
with a DNA of the present invention and purifying the antibodies by
conventional methods that are routinely used by those skilled in the art.

[0103] The present invention also provides host organisms that harbor a
vector carrying a nucleic acid encoding an antibody of the present
invention. The host organisms of the present invention are useful in
producing recombinant antibodies. The host organisms of the present
invention include goats. For example, transgenic goats of the present
invention can be created by the method described below. Specifically, a
fusion gene is constructed by inserting an antibody gene in frame within
a gene encoding a protein (goat casein or such) intrinsically produced in
milk. DNA fragments comprising the fusion gene which contains the
inserted antibody gene are injected into goat embryos, and the resulting
embryos are introduced into female goats. The antibodies of the present
invention can be prepared from milk produced by transgenic animals born
by the goats that received the embryos, or produced from progenies of
these animals. Hormones can be given to the transgenic goats to increase
the amount of milk containing the antibodies of the present invention
produced by the goats (Ebert, K. M. et al., Bio/Technology 12, 699-702
(1994)).

[0104] The present invention provides imaging agents for visualizing
arteriosclerotic sites, which contain an antibody that binds to the
oxidized LDL/β2GPI complex. The present invention also provides
imaging methods for visualizing arteriosclerotic sites, which comprise a
step of administering an antibody of the present invention that binds to
the oxidized LDL/β2GPI complex to mammals. The imaging agents
of the present invention are administered to mammals to visualize
arteriosclerotic sites. Such mammals include humans and nonhuman mammals
(for example, mice, rats, hamsters, rabbits, pigs, and monkeys). The
imaging agents of the present invention are useful in diagnosing
arteriosclerosis. The imaging agents of the present invention can be used
both in vivo and in vitro.

[0105] Arteriosclerotic symptoms are roughly divided into atheroma and
calcified lesion. Atheroma sites in arteriosclerosis are especially
stained by the imaging agents of the present invention.

[0106] Atheroma is a pathological condition of arteriosclerosis.
Macrophages are known to specifically take up via receptor oxidized LDL
which contains a large amount of cholesterol, and thereby become foamy.
The foamy macrophages accumulate and form plaques (atheromas) on the
intima of a blood vessel.

[0107] The imaging agent of the present invention is prepared by linking
an imaging label or probe to an antibody that binds to the oxidized
LDL/β2GPI complex, in particular, preferably antibody 3H3. The
imaging label or probe enables direct or indirect monitoring.

[0108] After in vivo administration (for example, intravenous
administration) of an above-described probe, the distribution or amount
accumulated can be assessed using an imagining device such as PET, SPECT,
or CCD camera.

[0109] Furthermore, in recent years, computer-aided tomography (computed
tomography; "CT" also refers to computed tomography) has been applied in
a clinical setting such as for disease diagnosis. Computer-aided
tomography is a technology for generating an image of the interior of an
object by scanning the object using a source of penetrating radiation,
and processing the data in a computer.

[0110] The CT technology is a technique for obtaining two-dimensional
cross sectional patterns of objects (cross sections, etc.) by recording
sectional images with positron emission tomography (PET), single photon
emission computed tomography (SPECT), magnetic resonance imaging (MRI),
or the like. These examination techniques are often used not only for
obtaining sectional images, but also for presenting three dimensional
graphic images by integrating the two-dimensional images using the
advanced computer-assisted image processing technology. Thus, the
examination techniques are powerful tools for specifying the
three-dimensional location of lesions, diagnosis, decision of operative
strategy, and so on.

[0111] For example, simple CT is used to obtain images by irradiating X
ray or the like without using any contrast agent. Tissue edema,
morphological abnormalities of bone, morphologies, and the like can be
observed without using any contrast agent. Meanwhile, enhanced CT refers
to CT in which images are taken after a contrast agent or the like having
high X-ray absorption is injected into a blood vessel. Enhanced CT can be
used to observe morphologies of blood vessels and tissues rich in blood
flow. Furthermore, the so-called next-generation CT has been developed,
and it can be used alone or in combination to detect the imaging agents
of the present invention. Such next-generation CT is not particularly
limited, and includes, for example, helical CT in which the irradiation
source moves in a spiral manner, and multi-detector computed tomography
(MDCT) (also referred to as multi-slice CT (MSCT)) in which detectors are
arranged in multiple rows in the direction of body axis.

[0112] When the labeled imaging probe (an imaging agent of the present
invention) is a radionuclide with high X-ray absorption, CT can be used
alone as a detector.

[0114] Such labeling substances include fluorescent labels, those using
enzymatic luminescence (luciferase), and those using fluorescence
(fluorescent proteins such as GFP, DsRed, and Kusabira Orange; and
fluorescent low-molecular-weight substances such as, FITC, Cy5.5, and
Alexa Fluor 750).

[0115] When enzymatic luminescence (luciferase) is used, it is necessary
to administer a substrate separately.

[0116] In particular, labels that have reduced influence from the animal's
intrinsic fluorescence are preferred, and labels that emit a signal with
high skin permeability are more preferred.

[0117] Magnetic resonance imaging (MRI), PET, and SPECT are used as an
imaging detector. In particular, when fluorescent probes are used, CCD
camera is preferably used as the monitoring device in terms of low
invasiveness.

[0118] For this reason, labels that emit light at a wavelength detectable
by CCD camera, for example, about 350 to 900 nm, are preferred.
Furthermore, devices that can be used to determine the intensity of light
source inside the body based on values obtained by monitoring the body
surface of a test animal with a CCD camera are preferred. When
fluorescent labels are used, the image may be a reflection fluorescence
image or transmission fluorescence image; however, it is preferable to
capture both images. Furthermore, the fluorescence images can be observed
three-dimensionally by superimposing multi-directionally recorded
fluorescence images (regardless of reflection or transmission) and
integrating information of the radiation source into the superimposed
images. This processing is preferred because it enables reproduction of
accurate three-dimensional locations and distribution. The
three-dimensional images obtained by this method can also be further
superimposed with CT images.

[0119] When the labeled imaging probe is linked to a radionuclide with
high X-ray absorption, CT can be used alone as the imaging detector (for
example, PET or SPECT) as described above, and can also be used to
determine the site, accumulated amount, and distribution of
arteriosclerotic plaques.

[0120] Alternatively, following in vivo administration (for example,
intravenous administration) of the-above described labeled imaging probe,
the labeled probe may be observed by CT alone or in combination with CCD.
When CT is used in combination with CCD, for example, a CCD image of
fluorescently labeled probe is superimposed with an image of simple CT
(and/or an image of enhanced CT). Specifically, CT images resulting from
simple-CT image extraction of organs such as bones and lungs (and/or
enhanced-CT image extraction of blood vessels and tissues) are integrated
with fluorescent probe images of major arterial lesions such as in the
heart. This enables more accurate understanding of the site, accumulated
amount, and distribution of arteriosclerotic plaques, three-dimensional
positional relationships relative to tissues and blood vessels, and
accurate three-dimensional images (localization) of arteriosclerotic
plaques.

[0121] The imaging agents of the present invention can be formulated, in
addition to the antibodies, with pharmaceutically acceptable carriers by
known methods. For example, the agents can be used parenterally, when the
antibodies are formulated in a sterile solution or suspension for
injection using water or any other pharmaceutically acceptable liquid.
For example, the agents can be formulated by appropriately combining the
antibodies of the present invention with pharmaceutically acceptable
carriers or media, specifically, sterile water or physiological saline,
vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers,
flavoring agents, excipients, vehicles, preservatives, binding agents,
and such, by mixing them at a unit dose and form required by generally
accepted pharmaceutical practices. The content of active ingredient in
such a formulation is adjusted so as to contain an appropriate dose
within the specified range.

[0122] Sterile compositions for injection can be formulated using vehicles
such as distilled water for injection, according to standard formulation
protocols.

[0123] Aqueous solutions to be used for injection include, for example,
physiological saline and isotonic solutions containing glucose or other
adjuvants such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride.
They may be used in combination with suitable solubilizers such as
alcohol, specifically ethanol, polyalcohols such as propylene glycol and
polyethylene glycol, and non-ionic surfactants such as Polysorbate 80®
and HCO-50.

[0124] Oils include sesame oils and soybean oils, and can be combined with
solubilizers such as benzyl benzoate or benzyl alcohol. They may also be
formulated with buffers, for example, phosphate buffer or sodium acetate
buffer; analgesics, for example, procaine hydrochloride; stabilizers, for
example, benzyl alcohol or phenol; or antioxidants. The prepared
injections are typically aliquoted into appropriate ampules.

[0125] The administration is preferably parenteral, and specifically
includes injection, intranasal administration, intrapulmonary
administration, and percutaneous administration. For example, injections
can be administered systemically or locally by intravenous injection,
intramuscular injection, intraperitoneal injection, or subcutaneous
injection.

[0126] Furthermore, the method of administration can be appropriately
selected depending on the patient's age and symptoms. The dosage of the
imaging agents of the present invention can be selected, for example,
from the range of 0.0001 to 1,000 mg per kg of body weight for each
administration. Alternatively, the dosage may be, for example, in the
range of 0.001 to 100,000 mg/person. However, the dosage is not limited
to these values.

[0127] The dose and method of administration vary depending on the
subject's body weight, age, symptoms, and intensity of fluorescent
labeling per mg antibody/sensitivity of detection device, and can be
appropriately selected by those skilled in the art.

[0128] The present invention also provides imaging kits for visualizing
arteriosclerotic sites, which comprise an antibody of the present
invention which binds to the oxidized LDL/β2GPI complex. The
kits of the present invention visualize arteriosclerotic sites when
administered to subjects. The above-described kits contain in addition to
an antibody of the present invention, for example, injectors (apparatuses
for drip infusion), adjuvants for suppressing non-specific adsorption
(for example, albumin), and such, without limitation thereto.

[0129] The kits may also contain items generally contained in kits, such
as instruction manuals, appropriate containers, and control reagents used
in imaging.

[0130] The present invention provides methods of screening for candidate
compounds as therapeutic agent for treating arteriosclerosis, which
comprise the steps of:

[0131] (a) administering to a nonhuman animal model of arteriosclerosis a
candidate compound and an antibody of the present invention which binds
to an oxidized LDL/O2GPI complex, for example, administering a
candidate compound to a nonhuman animal model of arteriosclerosis that
has been administered with an antibody of the present invention which
binds to the oxidized LDL/β2GPI complex,

[0132] (b) visualizing arteriosclerotic plaques in a nonhuman animal model
of arteriosclerosis administered with the antibody and candidate
compound, and in a nonhuman animal model of arteriosclerosis administered
with the antibody but not with the candidate compound;

[0133] (c) comparing arteriosclerotic plaques (for example, the size or
site of arteriosclerotic plaques) between a nonhuman animal model of
arteriosclerosis administered with the antibody and candidate compound
and a nonhuman animal model of arteriosclerosis administered with the
antibody but not with the candidate compound; and

[0134] (d) selecting a candidate compound that reduces or eliminates
arteriosclerotic plaques in a nonhuman animal model of arteriosclerosis
administered with the antibody and candidate compound as compared to a
nonhuman animal model of arteriosclerosis administered with the antibody
but not with the candidate compound.

[0135] Each step is performed using known techniques or techniques
described above.

[0136] Candidate compounds that can be used in the screening methods of
the present invention include, but are not limited to, purified proteins
(including antibodies), expression products of gene libraries, synthetic
peptide libraries, DNA and RNA libraries (including functional nucleic
acids such as aptamers and siRNAs), cell extracts, cell culture
supernatants, and synthetic low-molecular-weight compound libraries.

[0137] Nonhuman animal models of disease that can be used in the screening
methods of the present invention include, but are not limited to, mice,
hamsters, rats, rabbits, pigs, and monkeys.

[0138] Arteriosclerosis model mice include, for example, transgenic mice
in which a gene is overexpressed, and knockout mice that are deficient in
a gene as a result of gene targeting. Arteriosclerosis models include,
for example, apoE-deficient (apoE.sup.-/-) model (apoE (apolipoprotein E)
is a protein that forms LDL which is known as bad cholesterol), LDL
receptor-deficient (LDLR.sup.-/-) model, model introduced with human
apoB, and model introduced with dominant apoE mutation. Such model mice
also include type 2 diabetes model mice (KKAy), and arteriosclerosis
model mice which are produced by feeding C57BL6 mice with a high
cholesterol diet or such. The C57BL6 line is known to have the greatest
tendency of developing arteriosclerosis among mice, and mice of this line
sometimes show arteriosclerotic plaques by simply feeding on a high
cholesterol diet.

[0139] Arteriosclerotic plaques are sometimes seen in rabbits fed a high
cholesterol diet for about 2.5 months. Furthermore, LDL
receptor-deficient arteriosclerosis model rabbits include WHHL rabbits.

[0140] A pig arteriosclerosis model is also known, which has a tendency to
develop arteriosclerosis due to abnormality in the amino acid sequence of
the LDL receptor-binding domain of apoB. Those skilled in the art can
prepare arteriosclerosis model animals by referring to documents such as
"Kessensho/Doumyakukoka Model Doubutu Sakuseihou (Methods for producing
thrombosis/arteriosclerosis model animals), Ed., Koji Suzuki (Kinpodo)".
The resulting model animals can be used in the present invention.

[0141] Compounds that reduce or eliminate arteriosclerotic plaques, which
are selected by the screening methods of the present invention, are
candidate compounds of therapeutic agents for arteriosclerosis. Thus, the
present invention provides therapeutic agents for arteriosclerosis, which
comprise as an active ingredient a substance selected by the screening
methods of the present invention. The present invention also relates to
the use of compounds selected by the screening methods of the present
invention in manufacturing therapeutic agents for arteriosclerosis. When
substances isolated by the screening methods of the present invention are
used as a therapeutic agent, they can be used after they are formulated
using known pharmaceutical production methods. For example, such
substances are administered to patients in combination with
pharmaceutically acceptable carriers or media (physiological saline,
vegetable oils, emulsifiers, detergents, stabilizers, etc.). The
substance is administered transdermally, nasally, transbronchially,
intramuscularly, intravenously, or orally according to its properties.
The dosage depends on the patient's age, weight, and symptoms, and the
method of administration. However, those skilled in the art can select an
appropriate dose.

[0142] The nucleotide and amino acid sequences of the antibodies described
herein are shown in the Sequence Listing according to the SEQ IDs shown
below.

[0153] All prior art documents cited in the specification are incorporated
herein by reference.

EXAMPLES

[0154] Hereinbelow, the present invention will be specifically described
with reference to the Examples, but is not to be construed as being
limited to the illustrative embodiments described in the Examples.

[0156] 0.2 mg/ml oxidized LDL described above was incubated at a final
concentration of 0.2 mg/ml with human β2GPI (purchased from
Affinity Biologicals) at 37° C. for 16 hours to form the oxidized
LDL/β2GPI complex.

Example 2

Immunization with Antigen

[0157] Purified protein of human oxidized LDL/β2GPI complex was
mixed with the same amount of complete adjuvant (SIGMA; F5881). BALB/c
mice (female) were immunized through footpads with the resulting emulsion
at 5 to 50 μg/head every three to seven days several times. Three to
five days after the final immunization, inguinal lymph nodes were excised
from the mice, and fused with cells of mouse myeloma P3U1 (P3-X63Ag8U1).

Example 3

Cell Fusion, and Selection and Isolation of Monoclonal Antibody-Producing
Cells

[0158] Cell fusion was carried out based on the conventional method
described below. For every medium, fetal bovine serum (FBS) was used
after inactivation by incubation at 56° C. for 30 minutes. P3U1
was prepared by culturing in RPMI1640-10% FBS (containing penicillin and
streptomycin).

[0159] Cells from excised mouse inguinal lymph nodes were combined with
P3U1 at a ratio of 10:1 to 2:1. The mixed cells were centrifuged. As a
fusion enhancing agent, 50% polyethylene glycol 4000 (Merck; gas
chromatography grade PEG4000, Catalog No. 9727) was added little by
little to the precipitated cells while gently mixing to achieve cell
fusion. Then, RPMI1640 was added little by little to the mixture with
gentle mixing. The resulting mixture was centrifuged. The precipitated
fused cells were appropriately diluted with HAT medium containing 15%
FCS(RPMI1640, HAT-supplement (Invitrogen; 11067-030), penicillin, and
streptomycin), and plated at 200 μl/well in 96-well microplates.

[0160] The fused cells were cultured in a CO2 incubator (5% CO2,
37° C.). When the cells were sufficiently grown as colonies,
screening was carried out by sampling the culture supernatants.

[0161] In the screening, positive clones were selected by ELISA (described
in Example 4) using 96-well plates coated with the human oxidized
LDL/β2GPI complex, which was the same as that used as the
immunizing antigen. The clones were expanded using HT medium (RPMI1640,
HT-supplement (Invitrogen; 21060-017), penicillin, and streptomycin)
containing 15% FCS, and then cloned into single clones by the limiting
dilution method. This screening which used the anti-human oxidized
LDL/β2GPI complex antibody as an immunogen yielded seven types
of hybridoma clones including clone 3H3.

Example 4

Reactivity to Human Oxidized LDL/β2GPI complex and
β2GPI (ELISA)

[0162] The ELISA for detecting an anti-human oxidized LDL/β2GPI
complex antibody was carried out by the method described below.
Specifically, 50 μl of 1 μg/ml oxidized LDL/β2GPI was
added to each well of microplates (Nunc; Maxisorp). The plates were
incubated at 4° C. overnight to adsorb the complex, and then
blocked with 1% BSA. Antibody samples were diluted using an assay buffer
(1% BSA, 0.15 M NaCl/20 mM HEPES (pH 7.4)) to the antibody concentrations
indicated on the horizontal axis. 50 μl of each sample was added to
the wells, and the wells were incubated for 30 minutes. The solutions
were discarded, and the wells were washed with 0.1% Tween 20/PBS. Then,
50 μl of 2,000-times diluted HRP-labeled anti-mouse IgG (MBL code 330)
was added to each well of the plates, and incubated for 30 minutes. The
solutions were discarded, and the wells were washed with 0.1% Tween
20/PBS. Then, 50 μl of substrate TMB (MOSS; TMBZ) was added, and the
plates were incubated at room temperature for three minutes. After the
reaction was terminated by adding 50 μl of 0.18 M sulfuric acid,
detection was carried out using absorbance at 450 nm (FIG. 1A).

[0163] To detect the reactivity to β2GPI, ELISA was carried out
by the method described below. Specifically, 50 μl of 1 μg/ml
β2GPI was added to each well of microplates (Nunc; Maxisorp).
The plates were incubated at 4° C. overnight to adsorb
β2GPI, and then blocked with 1% BSA. Antibody samples were
diluted using the assay buffer (1% BSA, 0.15 M NaCl/20 mM HEPES (pH 7.4))
to the antibody concentrations indicated on the horizontal axis. 50 μl
of each sample was added to the wells, and incubated for 30 minutes. The
solutions were discarded, and the wells were washed with 0.1% Tween
20/PBS. Then, 50 μl of 2,000-times diluted HRP-labeled anti-mouse IgG
(MBL code 330) was added to each well of the plates, and incubated for 30
minutes. The solutions were discarded, and the wells were washed with
0.1% Tween 20/PBS. Then, 50 μl of substrate TMB (MOSS; TMBZ) was
added, and the plates were incubated at room temperature for three
minutes. After the reaction was terminated by adding 50 μA of 0.18 M
sulfuric acid, detection was carried out using absorbance at 450 nm (FIG.
1B).

[0164] Furthermore, various concentrations of β2GPI (up to 50
μg/ml) were prepared and added at 50 μl/well to microplates (Nunc;
Maxisorp). The plates were incubated at 4° C. overnight to adsorb
β2GPI. Then, the antibody reactivity was tested in the same
manner (data not shown).

[0165] The result showed that the reactivity towards the immobilized
oxidized LDL/β2GPI complex was: 2H6>3H3, 2A12, 3D4>4C12,
1H4. Alternatively, the reactivity towards the immobilized
β2GPI was: 2H6, 3D4>2A12, 4F10. 3H3 and 4C12 were not
reactive to the immobilized β2GPI (FIGS. 1A and B).

[0166] However, when the coating concentration in microtiter plates was
increased, 3H3 also exhibited reactivity (data not shown).

[0167] Next, as a method for assessing antibody reactivity, inhibition
test using a free antigen was carried out to evaluate the specificity of
each antibody.

[0168] In the reactivity assay (ELISA) for immobilized human oxidized
LDL/β2GPI complex and β2GPI, an inhibitory reaction
to immobilized antigen was carried out by having oxidized
LDL/β2GPI complex or β2GPI together when the
antibodies were added in the reaction (Schematic diagram of assay system
is shown in FIG. 2).

[0169] Specifically, 50 μl of 1 μg/ml β2GPI was added to
each well of microplates (Nunc; Maxisorp). The plates were incubated at
4° C. overnight to adsorb β2GPI, and then blocked with
1% BSA. Antibody samples were diluted to appropriate concentrations using
the assay buffer (1% BSA, 0.15 M NaCl/20 mM HEPES (pH 7.4)), and samples
of oxidized LDL/β2GPI complex or β2GPI, which serves
as a competitive antigen, were diluted to the antigen concentrations
indicated on the horizontal axis. 25 μl each of the diluted antibody
sample and antigen sample were added to the wells, and the wells were
incubated for 30 minutes. The solutions were discarded, and the wells
were washed with 0.1% Tween 20/PBS. Then, 50 μl of 2,000-times diluted
HRP-labeled anti-mouse IgG (MBL code 330) was added to each well of the
plates, and the plates were incubated for 30 minutes. The solutions were
discarded, and the wells were washed with 0.1% Tween 20/PBS. Then, 50
μl of substrate TMB (MOSS; TMBZ) was added, and the plates were
incubated at room temperature for three minutes. After the reaction was
terminated by adding 50 μl of 0.18 M sulfuric acid, detection was
carried out using absorbance at 450 nm.

[0170] The result showed that when the coexisting oxidized
LDL/β2GPI complex was the free antigen in ELISA, the binding of
3H3, 4C12, and 2A12 to immobilized oxidized LDL/β2GPI was
markedly inhibited, while β2GPI did not inhibit the binding. On
the other hand, the binding of 2H6 was inhibited when the free antigen
was the oxidized LDL/β2GPI complex, and the mixing with
β2GPI also inhibited the binding to some extent. As for 3D4,
stronger inhibition was observed with β2GPI than with oxidized
LDL/β2GPI complex as free antigen (FIG. 3).

[0171] From the results above, reactivity of antibodies can be summarized
as shown in Table 1 (Table 1 is shown in Example 7). 3H3 showed similar
reactivity to 4C12, but was not the same reactivity, and had different
specificity.

[0172] ApoE.sup.-/- mice and LDLR.sup.-/- mice (obtained from Jackson Lab,
and maintained in the animal experiment facility at Okayama University)
were fed a common diet (Oriental Yeast NMF) up to eight weeks old, and
then fed a high fat diet (common diet additionally containing 1%
cholesterol, 1% cholic acid, and 15% salt-free butter) for four to six
months. As a result, arteriosclerotic plaques developed, and thus
thickening and atheroma were observed in the thoracic or abdominal aorta.
Then, these eight-month-old mice were sacrificed. Cryosections of the
thoracic aorta, and aortic root and valves were prepared from the mice,
and observed as samples.

[0173] The prepared cryosections were fixed with paraformaldehyde and then
used in the experiment of fluorescent antibody immunostaining.

Labeling of Monoclonal Antibody with Cy5.5

[0174] Various monoclonal antibodies (1 mg/ml) were dialyzed against 0.1 M
carbonate buffer (pH 9.3) at 4° C. overnight, and each was
transferred into Fluorolink Cy5.5 monofunctional dye (1 tube). After 30
minutes of incubation at room temperature, the antibodies were treated
with a SephadexG-25 column to yield Cy5.5-labeled antibody.

Fluorescent Immunostaining of Cryosections

[0175] Sections were fixed with 1% paraformaldehyde for five minutes, and
then incubated with various monoclonal antibodies at 4° C.
overnight. After washing, the sections were incubated with an
FITC-labeled anti-mouse IgG or IgM antibody (secondary antibody) at room
temperature for one hour. Staining with DAPI and Rhodamine Phalloidin was
carried out by addition with the secondary antibody at the time of
incubation. Then, the sections were observed and photographed under a
fluorescent microscope.

Immunohistochemical Staining

[0176] The result showed that when used in fluorescent immunostaining of
C57BL6 mice fed a normal diet, both antibodies 3H3 and Mac3 stained
atheroma resulting from accumulation of foamy macrophages. 3H3 stained
the same areas (FIG. 4).

[0177] Fluorescent immunostaining of the aortic valve in
arteriosclerosis-prone model mice (apoE.sup.-/- fed a high fat diet) was
compared to the result obtained using different antibodies that recognize
the oxidized LDL/β2GPI complex. Antibodies positive for
atheroma in the staining were only antibodies 3H3 and A (FIG. 5).

[0180] Experiment 1: Cy5.5-labeled monoclonal antibody (0.25 mg/ml) was
administered at 0.15 ml/head via the caudal vein to apoE.sup.-/- mice fed
a high fat diet, which were prepared by the same method as described in
Example 6. The following three were administered: physiological saline
(PBS; control), Cy5.5-labeled antibody A, and Cy5.5-labeled antibody
3113. Twenty four hours after administration, the mice were photographed
alive for the full-body image after removing their thoracic skin (FIG.
7).

[0181] Experiment 2: Then, the heart intact with thoracic aorta was
excised and photographed (FIG. 7). The aortic root was intensely stained
by 3113 administration. Antibody A also stained to some extent; however,
the fluorescence intensity was weaker as compared to 3H3. There was no
stain with 2A12.

[0182] The fluorescence intensity was determined per unit area of the
aortic root. The fluorescence of PBS-administered control mouse was taken
as 1.0. When 3H3 was administered, fluorescence was three times stronger
than the control. There was no significant change in the fluorescence
intensity when other antibodies were administered (FIG. 10).

[0183] The specificity assessment of the antibodies described above is
summarized in Table 1.

[0184] β2GPI was added as an inhibitory, competitive antigen to
the immobilized oxidized LDL/β2GPI complex or immobilized
β2GPI, and the resulting inhibition was assessed by ELISA. The
result is as follows: in the case of immobilized oxidized
LDL/β2GPI complex, 3D4>2H6>4C12>3H3; and in the case
of immobilized β2GPI, 2H6>3D4 (4C12 and 3H3 bound only
weakly to immobilized β2GPI). 3H3 was highly specific to the
free (non-denatured) form of oxidized LDL/β2GPI complex in a
solution.

[0186] The antibody subclasses of the four clones are as follows: 3H3 and
4C12 are IgG2b; and 2H6 and 3D4 are IgG1.

Analysis of L-Chain Variable Region Gene

[0187] Hybridomas which produce four types of monoclonal antibodies (3H3,
4C12, 2H6, and 3D4) were each cultured in RPMI1640 supplemented with 10%
FCS. mRNAs were obtained from the hybridomas using the QuickPrep micro
mRNA purification kit (Amersham Biosciences; code 27-9255-01). The mRNAs
were converted into cDNAs using the First-Strand cDNA Synthesis kit
(Amersham Biosciences; code 27-9261-01). Gene amplification was achieved
by PCR using the cDNAs as a template. PCR was carried out using the 11
types of primer combinations listed below. The sequences of primers MKV1
to MKV11 were designed by analyzing the signal sequences of numerous
various monoclonal antibodies. Thus, the 11 types of primer sequences can
cover the L chain signal of almost every monoclonal antibody. An L-chain
variable region of interest is amplified by using at least a single PCR
pattern selected from 11 PCR patterns using combinations of the 11 types
of MKV primers with primer MKC which corresponds to the sequence of a
mouse L-chain constant region.

[0195] Hybridomas which produce four types of monoclonal antibodies (3H3,
4C12, 2H6, and 3D4) were each cultured in RPMI1640 supplemented with 10%
FCS. mRNAs were obtained from the hybridomas using the QuickPrep micro
mRNA purification kit (Amersham Biosciences; code 27-9255-01). The mRNAs
were converted into cDNAs using the First-Strand cDNA Synthesis kit
(Amersham Biosciences; code 27-9261-01). Amplification of H chain
variable region genes was achieved by PCR using the cDNAs as a template.
PCR was carried out using the 12 types of primer combination listed
below. The sequences of primers MHV1 to MHV12 were designed by analyzing
the signal sequences of numerous various monoclonal antibodies. Thus, the
12 types of primer sequences can cover the H chain signal of almost every
monoclonal antibody. An H-chain variable region of interest is amplified
by using at least a single PCR pattern selected from 12 PCR patterns
using combinations of the 12 types of MHV primers with primer MHCG2b or
MHCG1 which corresponds to the sequence of a mouse H-chain constant
region. Primer MHCG2b corresponds to the sequence of an H-chain constant
region of mouse IgG2b, while primer MHCG1 corresponds to the sequence of
an H-chain constant region of mouse IgG1. Thus, primer MHCG2b was used in
the PCR amplification of clones 3H3 and 4C12, which are of the IgG2b
subclass. Primer MHCG1 was used in the PCR amplification of clones 2H6
and 3D4, which are of the IgG1 subclass.

[0216] Fluorescence imaging was carried out using IVIS 200 Imaging System
(Xenogen) (for Cy5.5, [excitation, 640 nm; emission, 720 nm]; for Alexa
Fluor 750, [excitation, 745 nm; emission, 800 nm]). 0.25 mg/ml
Cy5.5-labeled antibody 3H3 (IgG) or 1.0 to 1.5 mg/ml Alexa Fluor
750-labeled antibody 3H3 was administered at 0.15 ml/head via the caudal
vein to ApoE.sup.-/- mice fed a high fat diet, and after two to 24 hours
under inhalation anesthesia, in vivo fluorescence was observed and
photographed using IVIS 200. The ApoE.sup.-/- mice were observed after
shaving, because their black hair absorbs fluorescence. First, the
fluorescence was observed with reflected light, and then with transmitted
light. Three-dimensional (3D) images of mice were generated and
integrated with the light source information (FIG. 9A: a
three-dimensional image by IVIS before integration). In the figure, red
dots correspond to fluorescent signals from labels linked to 3H3. The
denser red dots mean stronger fluorescence intensity, showing the
localization of the imaging agent.

Ex Vivo Imaging:

[0217] After 3D CT analysis, the mice were euthanized, and the hearts were
perfused with 10 ml of PBS. The hearts and aortae were excised and their
reflection fluorescence images were obtained using IVIS 200.

CT Imaging:

[0218] CT imaging was performed using eXplore Locus CT System (GE
Healthcare). Under inhalation anesthesia, the same mice used in the IVIS
200 imaging were irradiated with X ray to obtain CT images.

[0222] (B) IVIS 200 fluorescence image obtained using a specific antibody
(transmitted light; left) and CT image (middle) before integration, and
integrated image (right). In the fluorescence image (transmitted light;
left), as the red dots become denser, the fluorescence intensity becomes
stronger, suggesting that the imaging agent is localized and accumulated
at the position (site that exhibits stronger binding reactivity to 3H3).

[0224] The visible light is absorbed by the body while the light of
near-infrared wavelengths is hardly absorbed by the body. Thus,
near-infrared fluorescent labels are suitable for in vivo imaging. In
this experiment, antibodies labeled with Cy5.5 or Alexa Fluor 750 were
administered to mice via the caudal vein, and the resulting fluorescence
was monitored with IVIS 200 to assess the measurement conditions for the
reflection and transmission fluorescence. When ApoE.sup.-/- mice with
arteriosclerosis were observed by in vivo reflection fluorescence imaging
using a Cy5.5-labeled antibody, intense signals were found in the aortic
valve and thoracic aorta. Furthermore, by ex vivo imaging and ex vivo
fluorescence microscopy, the fluorescently labeled antibody administered
into the vein was demonstrated to be localized in arteriosclerotic
plaques. However, when a Cy5.5-labeled antibody was used, the signal of
transmission fluorescence was weak and thus it was difficult to identify
the site of fluorescence in the three-dimensional (3D) images. By
contrast, when an Alexa Fluor 750-labeled antibody was used, specific
intense signals were observed two hours after intravenous administration
in both reflection and transmission fluorescence images. In the generated
three-dimensional image, intense fluorescent signals were recognized in
the chest (FIGS. 8A and B, left panels). Then, the same mice were
photographed by CT. The image (FIG. 8B, middle panel) resulting from
extraction of bones and lungs from CT image was integrated with an IVIS
200 fluorescent image by Amira. The resulting integrated 3D image (FIG.
8B, right panel) showed that the presence of fluorescent signals in and
around the heart. In the figure, the denser red dots suggest stronger
fluorescence intensity, showing the localization of the imaging agent. CT
image (middle panel) and 3D-CT integrated image (FIG. 8B, right panel)
are shown. A three-dimensional image was generated as animation in a
computer-generated virtual space (three-dimensional graphic animation).
The sites labeled were observed from multiple angles (FIG. 8C).

[0225] The experimental result described above showed that when
ApoE.sup.-/- mice with arteriosclerosis were observed by in vivo
reflection fluorescence imaging using fluorescently labeled antibody 3H3,
intense signals were found in the aortic valve and thoracic aorta.
Furthermore, by ex vivo imaging and ex vivo fluorescence microscopy, the
fluorescently labeled antibody administered into the vein was
demonstrated to be localized in arteriosclerotic plaques.

[0226] The experiment described above demonstrated not only that
arteriosclerosis in mice could be visualized by using a near-infrared
fluorescent substance (Cy5.5 or Alexa 750)-labeled antibody, but also
that the images could be integrated with three-dimensional CT images.
Furthermore, it has been demonstrated that such antibodies enable
detection of human arteriosclerotic plaques. The experimental results
described herein will lead to clinically applicable technologies for
diagnostic imaging. In addition, the mouse imaging techniques are already
practicable as a screening system for drug discovery.

INDUSTRIAL APPLICABILITY

[0227] The sites (locations) of arteriosclerosis cannot be identified by
conventional tests for arteriosclerosis. In contrast, the present
invention provides non-invasive diagnostic methods that allow visual
identification of the site and size of arteriosclerotic plaques (in
particular, atheroma and atherosclerosis).

[0228] A screening system for therapeutic agents to treat atherosclerosis
can be constructed by using arteriosclerosis-prone model mice (for
example, apoE-deficient (ApoE-/-) mice; which maintain high plasma
cholesterol level, and spontaneously develop an atherosclerosis-like
condition) and antibodies for the imaging.

[0229] Furthermore, an imaging system for clinical diagnosis can be
constructed by converting the antibodies into humanized antibodies. Thus,
plaques or such detached from atheroma lesions of arteries are known to
cause arterial embolism which leads to cerebral embolism or myocardial
infarction. Methods for monitoring human arthrosclerosis which progresses
insidiously, asymptomatically, and chronically can be expected to benefit
strategies for preventing or treating lifestyle-related diseases.